A novel, universal method for measuring enzymatic activity using micro-calorimetry (Todd & Gomez, 1997) is applied to the E. coli chaperonin GroEL. Biocalorimetric assays measure the rate of heat evolution during conversion of substrate to product. Calorimetry thus gives a direct measurement of the rate of an enzymatic reaction, as opposed to conventional assays where rate is calculated as a derivative of a quantity with time. Isothermal calorimetric analysis of GroEL ATPase activity allows high-resolution rate analysis of turnover. In the presence of ATP regenerating system, the kinetics were biphasic (i.e., the kinetics of ATP hydrolysis by the two toroids can be distinguished). One of the two rings is highly susceptible to product inhibition, thus in the absence of a regenerating system, turnover by only one of the two rings is observed. The effects of ionic cofactors K+ and Mg2+, and unfolded proteins (cam a-lac) were also studied. The kinetics of the two rings become indistinguishable at high [K+], whereas the kinetics are well-separated at lower concentrations. GroEL can bind up to two unfolded proteins (one on each ring). The ATPase activity with one or two unfolded proteins bound is examined.